1. Field of the Invention
This invention relates to the isolation of the LCB1 and LCB2 genes of the year Saccharomyces cervisiae that encode subunits of the enzyme serine palmitoyltransferase (SPT), the first enzyme leading to synthesis of the long-base component of sphingolipids. The invention further relates to method of using these genes to either inhibit SPT activity or to inhibit synthesis of the enzyme. Furthermore, the invention relates to methods for construction strains of S. cervisiae or other organisms that can be used to select and test for compounds that either inhibit SPT activity or to inhibit synthesis of the enzyme.
2. Description of the Background
Sphingolipids are abundant in the membranes of fungi (Brennah, P. J., & Losel, D. M. 1978. Fungal lipids, in Microbial Physiology, Rose, A. H. & Morris, P. G., Eds. 17, 47-179, Acad. Press., N.Y.), animals (Seeley, C. C. and Siddiqui, B. 1977; the Glycojungates, Horowitz, M. I. and Pigman, W., eds., Acad. Press, N.Y. 1:495), and higher plants (Laine, R. a., Hsieh, T. C.-Y., & Lester, R. L. Glycophosphoceramides from plants, in Cell Surface Glycolipids, p.65, Am. Chem. Soc. Symp. Ser. 128, Am. Chem. Soc. Wash, D.C.) 1980. In spite of much effort, it has been difficult to understand the exact biological role(s) of sphingolipids and their mode of action at the molecular level. In animals, sphingolipids are thought to play a role in such general cellular events as cell-to-cell recognition, regulation of cell growth, and differentiation. The prevalence of sphingolipids suggests that they play vital roles in cells and direct proof that sphingolipids are essential cellular components has been obtained with the discovery of mutants of S. cervisiae that absolutely require a sphingolipid long-chain base (see below) for growth (Wells, G. B. and Lester, R. L.; J.Biol. Chem. 258: pages 10200-10203 (1983)) and viability (Pinto, W. J., Wells, G. B., Williams, A. C., Anderson, K. A., Teater, E. C., and Lester, R. L., Fed. Proc. 45: 1826 (1986)).
sphingolipids are derivatives of ceramides containing sugars and sometimes phosphates. Ceramides usually contain a fatty acid of 20-26 carbons connected via an amide linkage to a long-chain base. The major long-chain bases and their predominant distribution are: ##STR1##
Reaction (a), the first committed step in sphingolipid biosynthesis (reviewed in Merrill, A. H. and Jones, D. D. 1990. Biochemica et Biophysica Acta. 1044:1-12) is catalyzed by serine palmitoyltransferase (SPT, also called 3-ketodihydrosphingosine synthetase). This enzyme has been shown to occur in the fungus Hansenula ciferri (Snell, E. E., Di Mari, S. J., and Brady, R. N. 1970. Chem. Phys. Lipids, 5:116-138), in beef liver (Stoffel, W. 1970. Chem. Phys. Lipids. 5:139-158), and in the bacterium Bacteroides melaninogenicus (Lev, M., and Milford, A. F. 1973. Arch. Biochem. Biophys. 157:500-508). Other evidence for this reaction comes from our own work in S. cervisiae (Pinto et al., 1986; Pinto W. J., Wells, G. W. and Lester, R. L. 1992. J. Bacteriol. 174:2575-2581). The enzyme has never been purified to homogenity and characterized in any detail (reviewed in Merrill, A. H. and Jones, D. D. 1990. Biochemica et Biophysica Acta. 1044:1-12).
In reaction (d) the long-chain base is attached to a fatty acid to form a ceramide. In all organisms ceramides are converted to complex derivatives, the sphingolipids, by the addition of polar groups to the 1-hydroxyl. The sphingolipids in animals contain various oligosaccharides inked glycosidically to the ceramide to yield glycosphingolipids and also contain choline linked by a phosphodiester bond to ceramide to yield the abundant compound sphingomyelin. Certain sphingolipids in fungi and plants differ from the sphingolipids in animals because the 1-hydroxyl is linked through a phosphoryl group to inositol (myo-inositol) rather than directly to a sugar. This core structure, inositol-phosphorylceramide, or inositol-P-ceramide ("IPC", Smith, S. W., and Lester, R. L. 1974. J. Biol. Chem. 249:3395-3405), along with mannose-inositol-P-ceramide, (MIPC, ibid) and mannose-(inositol-P).sub.2 -ceramide (M(IP).sub.2 C, (Steiner, S., Smith, S. Waechter, C. J., and Lester, R. L. 1969. Proc. Natl. Acad. Sci. U.S.A. 64:1042-1048) collectively constitute the sphingolipids in S. cervisiae (Smith, S. W., and Lester, R. L. 1974. J. Biol. Chem. 249:3395-3405). Phosphoinositol sphingolipids are also a major class of lipids in plants (for references see Kaul, K. and Lester, R. L. 1975. Plant Physiol., 55:120-129) and parasites (Singh, B. N., Costello, C. E., and Beach, D. H. 1991. Arch. Biochem. Biophys. 286:409-418).
Because sphingolipids are vital for S. cerevisiae, the long-chain base biosynthesis pathway would appear to be a good target for antifungal compounds. In fact, sphingolipids may be vital for all organisms that contain them, and therefore, any compound that would inhibit long-chain base biosynthesis might inhibit growth of an organism that contained sphingolipids.
Accordingly, there is a need to begin to identify or design such inhibitory antifungal compounds to target the long-chain base biosynthesis pathway, which would appear to be a good target for antifungal compounds. Therefore we isolated two S. cerevisiae genes, LCB1 (SEQ ID NOS.: 1-3) and LCB2 (SEQ ID NOS.: 4-6), that most likely encode subunits of SPT. These are the first genes involved in long-chain base biosynthesis to be isolated from any organism. The genes provide a unique opportunity to identify compounds that block SPT activity or synthesis in specific organisms.